The term “food poisoning” generally brings to mind bacterial food poisoning caused by bacteria such as Salmonella, Vibrio parahaemolyticus, and pathogenic E. coli, or natural toxin food poisoning caused by natural toxins contained in, for example, globefish or mushrooms. In addition, a very large number of food poisoning cases are caused by viruses, such as Norwalk-like viruses (hereinafter referred to as NLVs), rotavirus, astrovirus, enterovirus, adenovirus, and hepatitis A virus. Recent epidemiological research has revealed that, among other viruses, Norwalk-like viruses are typical food-poisoning viruses.
Norwalk virus was first identified in 1972 after an outbreak of gastrointestinal illness in the U.S.A. Under an electron microscope, the virus is observed as a small spherical virus of about 30 nm in diameter having an unclear surface structure, and since then viruses having similar shapes have been collectively called “small round structured viruses” (SRSVs). In the meantime, in 1974, calicivirus, which had been well known in veterinary medicine and which measures about 30 nm in diameter and assumes a unique surface structure resembling a “Star of David,” was first identified in a human patient; specifically, in a patient suffering winter vomiting disease, which was at that time epidemic in Britain. Since then, viruses having a shape similar to the above have been called classical human caliciviruses.
These viruses are very difficult to grow in tissue culture cells or in experimental animals, and therefore, for some time the only feasible method was to isolate and culture the viruses on volunteers by use of stool specimens. Thus, characterization of the viruses was quite difficult. In 1990, a research group led by X. Jiang cloned the genome of Norwalk virus, and since then, gene analysis of these viruses has been energetically performed. Such efforts have revealed that an SRSV and a classical human calicivirus both belong to the family of Caliciviridae, having a single stranded “plus” RNA (plus-stranded). In the XIth International Congress of Virology, the family Caliciviridae was reported to comprise four different genera.
Thus, a group of viruses that had been called SRSVs was determined to belong to the genus Norwalk-like viruses (NLVs), and another group that had been called classical human caliciviruses was determined to belong to the genus Sapporo-like viruses (SLVs). Moreover, from an accumulation of data of genomic nucleotide sequences of viruses collected from a vast number of clinical specimens, NLVs have been confirmed to be classified into two genogroups I and II; i.e., genogroup I (GI) encompassing Norwalk viruses and Southampton viruses, and similar viruses; and genogroup II (GII) encompassing Hawaii viruses, Snow Mountain viruses, and similar viruses.
NLV infections in humans primarily occur by the mediation of foods (fish, shellfish, and water). Most of the viral food-poisoning cases that frequently occur during winter are believed to be caused by ingestion of shellfish such as oysters, and in fact, in a great number of study reports, oysters are identified as the source of infection with NLtVs. Some reports describe that ingestion of a sandwich contaminated with NLVs caused infection. Thus, presumably, NLV infection readily spreads through feces from an infected patient. (This virus is known to have strong infectivity and to cause infection even in a case where several to about one hundred viruses are present in a food product).
Once food poisoning has occurred, needless to say, identification of the cause and the contamination source is a critical issue. That is, the food-poisoning patients must be treated as quickly as possible through appropriate selection of a therapeutic method, which would be realized by identifying the cause of the food poisoning, and simultaneously, spreading of food poisoning must be stopped by identifying the contamination source as early as possible.
In particular, in order to identify the cause and the contamination source of food poisoning caused by pathogenic microorganisms, the following are required: detection and identification of the pathogenic microorganism that caused the illness (i.e., identification of the cause of the food poisoning); and identification of the food and the food manufacturing facility that caused the food poisoning, on the basis of, for example, the diet history of the patient suffering food poisoning (identification of the contamination source of the food poisoning).
Conventionally, an electron microscope has been employed to detect the above-mentioned NLVs. However, methods employing an electron microscope require an intricate procedure, and in addition, rapid and accurate detection of viruses is difficult in cases where the quantity of the viruses is small. In particular, since a very small amount of NLV particles exhibit infectivity, rapid and accurate detection of NLVs in, for example, contaminated foods is keenly desired, and yet, realization has been difficult Moreover, detection methods employing an electron microscope require a large facility for accommodating the electron microscope, and thus, detection through electron microscopy has been possible in only a limited number of facilities.
By keeping pace with the recent progress in gene analysis techniques, more sensitive, more rapid gene analysis through RT-PCR has now been performed frequently. In order to detect a virus through use of this method, primers for amplifying a specific region of the gene (hereinafter referred to as gene amplification primers) and primers used in the process of detecting a gene amplification product of interest on the basis of the presence of the specific region serving as an index (hereinafter referred to as detection primers) must be designed and employed. Particularly in the case of viruses such as NLVs, design of such primers encounters a problem which is very difficult to solve. That is, viruses easily undergo mutation and therefore, even in the case in which the virus responsible for the previous outbreak of food poisoning falls within the same group of the virus that is now epidemic, there is a high risk that detection may be disabled unless primers different from those employed for detection of the virus in previous outbreaks are used for the current food poisoning. Needless to say, attaining accurate identification of the source of contamination will still require use of detection primers each individually specific to viral mutation variants. However, this would only be required for the purpose of verification and would suffice if performed after identification of the causative virus of the food poisoning and identification of the source of infection is almost complete. More importantly, rapid identification should be given a high priority so as to establish a therapeutic regimen for the food-poisoning patient and to prevent spreading of contamination.
In order to solve the above problem, a need exists for discovering a highly conserved region in genes of a virus of interest, and designing, among other things, detection primers which correspond to the region, thus providing means for detecting the virus through use of such tools.
Accordingly, an object of the present invention is to identify a highly conserved region in genes of NLVS, and, on the basis of the information thus obtained, to provide rapid, accurate means for detecting NLVs.